BRPI0616439A2 - amphiphilic polymer complex-pdgf, process of preparation and use thereof, therapeutic composition, and use thereof - Google Patents

Abstract

Amphiphilic Polymer-PDGF COMPLEX, PREPARATION AND USE PROCESS, THERAPEUTIC COMPOSITION, AND USE OF THEM. The present invention relates to novel Platelet-Derived Growth Factor (PDGF) complexes associated with amphiphilic polymers enabling improved in vitro and in vivo physical and chemical stability of the therapeutic protein for pharmaceutical applications. It also relates to the process of preparing the amphiphilic polymer-PDGF complex wherein the polymer / PDGF-BB complex is prepared in aqueous medium and in the absence of an organic solvent capable of denaturing the protein and the use of said amphiphilic polymer complex. PDGF for the preparation of a healing therapeutic composition for the treatment of topical ulcers.

Description

"Amphiphilic Polymer-PDGF COMPLEX, PROCESS FOR THE PREPARATION AND USE OF THEM, THERAPEUTIC COMPOSITION, AND USE OF THEM"

The present invention relates to novel Platelet-Derived Growth Factor (PDGF) Platelet-Derived GrowthFactor complexes associated with amphiphilic polymers enabling improved in vitro and in vivo physical and chemical stability of therapeutic protein for pharmaceutical applications.

PDGFs are glycoproteins of about 30,000 Daltons, composed of two polypeptide chains linked together by two disulfide bridges. Four types of chains have been identified A 1, C and D. The native protein exists as an AB type homodimer or heterodimer (Oefner C. EMBO J. 11, 3921-2926, 1992).

PDGF was first isolated on platelets. These growth factors are released upon blood coagulation, capable of promoting the growth of different cell types (Ross R. et al., Proc. Natl. Acad. Sci. USA, 1974, 71, 1207; Kohler N. & Lipton A. , Exp. CellRes., 1974, 87, 297). PDGF is now known to be produced by a number of different platelet cells and is mitogenic to most mesenchymal-derived cells, i.e. blood, muscle, bone and cartilage cells, as well as tipoconjunctive cells (Raines EW, in "Biology of Platelet-Derivated Growth Factor", 1993, Westermark, B. et C. Sorg, Ed. Basel, Kerger, p. 74). Numerous articles also tend to demonstrate that macrophage PDGF behaves as a chemotactic and mitogenic agent for smooth muscle cells, which contributes to the myointimal thickening of the arterial walls characteristic of arteriosclerosis (Ross R. et al., Science, 1990, 248, 1009). . PDGF activities, on the other hand, include notably stimulating the release of granules by neutrophil monocytes (Tzeng DY et al., Blood, 1985, 66, 179), facilitating steroid synthesis by Leydig cells (Risbridger GP, Mol. Cell Endocrinol. 1993, 97,125), the stimulation of neutrophil phagocytosis Wilson E. et al., Proc. Natl. Acad.Sei. USA, 1987, 84, 2213), modulation of detrombospondin expression and secretion (Majak RA et al., J. Biol. Chem., 1987, 262, 8821), and post-regulation of the ICAM-I gene in muscle cells. vascular lesions (Morisaki N. et al., Biochem. Biophys. Res. Commun., 1994, 200, 612).

Given these various properties, the use of recombinant PDGFs in the pharmaceutical domain has already been envisaged. The use of PDGF has been notably approved for the treatment of pelvic ulcers (Regranex, J&J) and for periodontal repair (GEM 21 S, Biomimetic).

Ulcer healing, such as skin healing in general, is a complex phenomenon that requires coordinated intervention at the time and space of numerous cell types, which can be summarized in three phases: an inflammation phase, a proliferation phase, and a remodeling phase.

In the inflammation phase, about 7 days for normal healing, macrophages kill bacteria, debride damaged tissues and regenerate tissues. For this, macrophages secrete collagenases, ascitocins and growth factors.

During the proliferation phase, from the third day to the third week for normal healing, three events follow. The wound comes from granulation tissue, angiogenesis develops, and the secondary wound of epithelial cells. Granulation tissue grows at the edges to the center. Fibroblasts abundantly produce type III collagen.

During remodeling, from the third week to 1 or even 2 years, granulation tissues mature, fibroblasts produce less collagen. Blood vessels formed during granulation that are uselessly eliminated by apoptosis. Type III collagen is replaced by type I collagen that is organized according to the tension and sericule lines.

In this process, PDGF plays a key role.

When a wound forms, platelets aggregate and release oPDGF. PDGF attracts neutrophils, macrophages and fibroblasts upon measurement and is a potent mitogenic. Macrophages, endothelial cells synthesize and in turn secrete PDGF. PDGF stimulates the production of extracellular matrix by fibroblasts, essentially non-collagen compounds such as glucosaminoglycans and adhesion proteins (J.Forton et al, Essential Practice of Surgery, Springer, 2003, chap. 7, 77-89).

Chronic wounds such as diabetic foot ulcers, venous ulcers and pressure ulcers have the particularity of healing very slowly and sometimes incompletely because the healing method does not develop normally (R. Lobmann et al, J. of Diabètesand its complications , 2006, 20, 329-335).

The healing process is in fact a delicate balance between a process of destruction needed in order to eliminate damaged tissues and the repair process that leads to the formation of newborns. Proteases and growth factors play a crucial role in regulating this balance. In the case of chronic wounds, this balance is broken in favor of degradation which explains its delayed healing. So since there are different types of chronic wounds, biochemically they are relatively similar in that they are characterized by prolonged phases of inflammation leading to high levels of proteases and thus decreasing the activity of the decreasing factors (G. Lauer et al. J. Invest. Dermatol. 115 (2000) 12-18). This degradation of growth factors contributes to an overall detained loss associated with chronic wounds not favoring healing. (D.R. Yager et al. J. Invest. Dermatol. 107 (1996) 743-748).

There is currently on the market a recombinant human PDGF-BB medicine corresponding to the international denomination "becaplermin" sold under the tradename Regranex®. This medicine is indicated for the treatment of diabetic lower limb ulcers. It is presented in the form of a gel with topical application and allows the healing of ulcers. It allows, particularly as endogenous PDGF, to favor cell proliferation and thus the formation of new tissues.

This treatment has limited efficacy. (Cullen et al. The International journal of biochemistry & Cell Biology 34,1544-1556,2002) even if clinical studies have demonstrated improvements in healing and healing required for healing (Greenhalgh et al., American Journal of Pathology, 136,1235-1246 1990 ; Ladin Plastic and Reconstructive Surgery, 105, 1230-1231 2000; Holloway et al. Wounds, 5 / 4,198-206; Mandracchiaet et al., Clinics in Pediatric Medical and Surgery, 18,189-209 2001; Wieman J. AmericanDianalofSurgery, 176,74S-79S 1998).

The Regranex product containing PDGF -BB marketed by J&J has been shown to be effective in increasing the healing rate to 50% of patients treated by only 36% for patients who received only standard wound treatment. Despite this significant improvement in the treatment of diabetic foot ulcer, it should be noted that only 50% of patients heal after long and costly treatment. In the case of non-healing, consequences can be extremely severe and in many cases lead to lower limb amputation. It is necessary to add that the average duration of treatment is very long, about 20 weeks and its application is costly and difficult due to a wound cleaning and Regranex application in the morning followed by 12 afterwards a wound cleaning. frequency of nursing care. In addition, the average cost of a week-long treatment is excessively high (around US $ 1400).

Partial efficacy may be explained by rapid PDGF degradation of the wound to be treated. This degradation results in the case of chronic injury from a prolonged state of inflammation generating, at the injury level, an environment hostile to PDGF by stimulation of overproduction deproteases.

Although degradation control is required for wound healing, excessive proteolytic activity is detrimental leading to extracellular matrix degradation (F. Grinnell et al. J. Invest.Dermatol. 106 (1996) 335-341 et CN. Rao et al. J. Invest. Dermatol. 105 (1995) 572-578) and molecules having a key functional role as growth factors (V. Falanga et al. J. Derm. Surg. One. 18 (1992) 604-606; DR Yager et al., Wound Rep. Reg. 5 (1997) 23-32 et al., Wlaschek et al.

Br. J. Dermatol. 137 (1997) 646-647). In fact, growth factors such as PDGF, TGF β or bFGF are key elements in the healing process due to their ability to induce cell migration, proliferation, protein synthesis, matrix formation and more generally control of the process. repair process. However, these growth factors are protein molecules, and consequently sensitive to proteolytic degradation. Several studies show that the degradation of growth factors such as PDGF is much faster while being placed in contact with fluids from chronic wounds because they contain high concentrations of metalloproteinases (DR Yager et al. J. Invest.Dermatol. 107 (1996) 743. -748).

For the treatment of Regranex venous ulcers in a pilot clinical study reported in the publication (TJ. Wieman, Wounds, 2003, vol15, no. 8, 257-264) only showed a small improvement in the actual treatments based on regular wound cleaning with a compression therapy.

The problem of PDGF instability for example was revealed when producing protein. PDGF is known to be particularly sensitive to post-translational proteolysis (Hart et al., Biochemistry 29: 166- 172, 1990 and US Patent 07/557 219) and notably at the level of binding between the amino acid position 79 and lysine deposition 80 or the bond between the arginine at position 27 and the arginine deposition 28 of the PDGF chain.

This proteolytic instability poses a major problem in the context of obtaining this protein which is recombinantly produced yeast according to the procedure described in US Patent 4,845,075. Indeed US Patent 7,084,262 teaches that the analysis and purification of PDGF-BB leads to the achievement of 21 isoforms resulting from post-translational endoproteolytic cleavages. This great structural heterogeneity consequently results in a 50% decrease in the activity of the genetically engineered protein in relation to the mature protein.

Thus, recent clinical results from Cardium following a press release dated August 14, 2006 (www.prnewswire.com) on unhealed diabetic foot ulcers after 14 weeks show the potential that PDGF-BB treatment may offer. . Cardium's proposed solution is to introduce the PDGF -BB expression gene into the wound cells to overexpress it locally. This gene therapy with the help of an adenovector allowed to heal close to 80% of these diabetic foot ulcers resistant to current treatments in a cohort of 15 patients. This therapeutic solution is promising. But pharmaceutical developments in gene therapy-based treatments are still very dangerous today for safety reasons linked to the use of adenovirus-like viral vectors.

Therefore, there is a need and a possibility for improved current diabetic foot ulcer treatments with PDGF.

In the case of treatment of diabetic foot ulcer, the objective of

last one is triple:

- accelerate healing,

- increase the cure rate,

- simplify the treatment protocol.

Note also the case of venous ulcers and depression ulcers, causes of severe pain and very serious medical complications.

The problem to be solved is therefore essentially a protection of PDGF over the chronic wound.

Several solutions have been proposed.

U.S. Patent 5,905,142 describes a means of remedying these PDGF-referenced proteolysis problems by generating protein mutants having increased resistance to proteolytic attacks by replacing or suppressing one or more amino acids lysine or arginine near potential cleavage sites. This strategy to make protein more resistant to proteases is not satisfactory. This genetic modification of PDGF may lead to modifications of biological activity with different affinities to different receptors which may also induce toxicological problems. Furthermore, such a modification of PDGF requires further pharmaceutical development, extremely costly and dangerous.

In the 1970s when this protein was widely studied, it was found that purification was extremely delicate because PDGF "is a very adherent protein" due to its cationic and hydrophobic properties (Heldin.CH EMBO J. 11: 4251-4259, 1992; Raines andRoss 257 (9): 5154-5160.1982; Antoniades, PNAS 78: 7314.1981; Deuel et al. J. Biol. Chem. 256: 8896 (1981). PDGF is, in effect, a strongly cationic protein whose isoelectric point is between 9.8 and 10.5. Other authors confirm this behavior as Wei etal (Dianal of controlled release 112: 103-110, 2006) who explain that PDGF is easily adsorbed onto the container surfaces in which it is found in solution. The authors solve the problem by adding a mixture of either 0.1% BSA or 0.1% BSA / Tween 20. Mixtures largely solve the problem because up to 95% of the protein can be found in solution. But these solutions are unsatisfactory from a pharmaceutical point of view given the animal origin of BSA and the risks associated with bovine spongiform encephalopathy.

Another solution proposed by the same authors is to add a more potent anionic surfactant (SDS) that allows keeping the PDGF in solution. Unfortunately, SDS also induces partial protein denaturation leading to a loss of bioactivity. This solution is therefore not satisfactory for stabilizing the protein.

In WO 93/08825, the inventors have shown that purified PDGF exhibits strong instability when formulated as a gel for topical application. They exemplify PDGF incompatibility with a number of products classically used to form pharmaceutical products such as methylcellulose or hydroxypropylcellulose as well as some conventional preservatives such as benzyl alcohol. The authors pose the problem by explaining that there is a need to formulate PDGF in the form of a gel for topical administration while having good long-term stability. The same authors show that PDGF in solution degrades by a neutral pH deamination process and that the protein is more stable to slightly acidic umpH. The authors show that by combining various parameters, a polymer showing no interactions with the protein, a slightly acid pH buffer limiting the deamination reaction and a neutral preservative against the protein. PDGF can be formulated to obtain a pharmaceutically stable formulation.

The authors show that it is possible to obtain a storage stable formulation by addition of a polymer without protein interactions under conditions of maintaining formation at a slightly acidic pH in order to avoid protein de-starch degradation reactions, however this solution is not satisfactory because It does not allow to protect the growth factor of proteolytic degradations in vivo at physiological pH.

In patent WO97 / 12601 which describes gel formulations of PDGF, the authors explain that the cellulose derivative they use is capable of stabilizing growth factors for eventual loss of activity upon storage. For this, they are based on the results previously obtained with PDGF in U.S. Patent 4,717,717. However, they also explain that the stability of the PDGF-containing cellulose gel can be greatly improved by adding to the formulation a charged chemical species such as charged amino acids or metal ions. Moreover, this solution allows to stabilize the growth factors in the formulation when storing the product but does not allow a stabilization of these growth factors against proteases present at the level of chronic wounds at physiological pH.

There is therefore a therapeutic interest to stabilize and protect growth factors, notably PDGF, in order to increase its effectiveness in treating chronic wounds and more particularly diabetic foot ulcers. The invention therefore relates to the stabilization of PDGF against chemical or physical degradation by intervening at physiological pH in vitro and in vivo for the elaboration of a complex between an amphiphilic polymer and a PDGF.

The invention therefore relates to the formation of a complex between an amphiphilic polymer and a PDGF (amphiphilic polymer-PDGF) complex providing a chemical and physical stabilization to the protein against degradation in physiological pH in vitro and in vivo.

The present invention therefore relates to a physically and chemically stable water-soluble amphiphilic PDGF complexopolymer, wherein:

The amphiphilic polymers are comprised of a hydrophilic skeletopolymeric functionalized by hydrophobic substituents and hydrophilic groups according to the following general formula.

Polymer

<formula> formula see original document page 11 </formula>

SD = m monomer units

• R, R 'being a bond or chain comprising from 1 to 18 carbons, optionally branched and / or unsaturated comprising one or more heteroatoms, such as O, N and / or S, R and R' being identical or different from each other.

• F, F 'being an ester, a thioester, an amide, a carbonate, a carbamate, an ether, a thioether, an amine, F and F1 being identical or different from each other

• X being a hydrophilic grouping which can be:

o a carboxylate

the one sulfate

the one sulfonate

o a phosphate a phosphonate

• Y being a hydrophilic group may be a sulfate

the one sulfonate

the one phosphate

the one phosphonate

• Hy being a hydrophobic grouping can be

o a C8 to C30 straight or branched alkyl optionally unsaturated and / or containing one or more heteroatoms such as O, N or S,

o a straight or branched C8 to C8 alkylaryl or arylalkyl, optionally unsaturated and / or possibly containing a heteroatom

o an unsaturated C8 to C30 polycycle,

η and o are between 1 and 3,

h represents the molar fraction of hydrophobic motif with respect to a monomeric unit comprised between 0,01 and 0,5,

χ represents the molar fraction of hydrophilic groups in relation to a monomeric unit comprised between 0 and 2.0,

y represents the molar fraction of hydrophilic groups with respect to a monomeric unit comprised between 0 and 0,5,

- PDGF is selected from the group consisting of PDGFs (Platelet-derived growth factors).

It refers to a characterized complex wherein PDGF is selected from the group consisting of recombinant human PDGFs bearing two B chains (rh PDGF -BB).

It refers to a characterized complex wherein the PDGF is PDGF-BB.

The substituents of the amphiphilic polymers are divided into controlled or statistical mode. Among the polymers having a controlled distribution of substituents, for example, block copolymers and alternate copolymers may be cited.

Statistical copolymer

<formula> formula see original document page 13 </formula>

Thus, in one embodiment, the invention also relates to an amphiphilic polymer-PDGF complex characterized in that the polymer is selected from the polymers whose substituents are statistically divided.

In one embodiment, the invention also relates to an amphiphilic polymer-PDGF complex characterized in that the amphiphilic polymer is selected from polyamino acids.

In one embodiment, the polyamino acids are selected from the group consisting of polyglutamates or polyaspartates.

In one embodiment, the polyamino acids are homopolyglutamates.

In one embodiment, the polyamino acids are homopolypartates.

In one embodiment, the polyamino acids are aspartate and glutamate copolymers. These copolymers are either in block or statistical form.

In one embodiment, the invention also relates to an amphiphilic polymer-PDGF complex characterized in that the polymer is selected from polysaccharides.

In one embodiment, polysaccharides are selected from the group consisting of hyaluronans, alginates, aschitosones, galacturonans, chondroitin sulfate, dextrates, asceluloses.

The group of celluloses is made up of acid-functionalized celluloses such as carboxymethyl cellulose.

The dextran group consists of dextrans functionalized by acids as carboxymethyl dextran.

In one embodiment, the polysaccharide is a soluble dextran derivative responding to the following formula (I)

DMCaBbSuc (I)

on what

D represents a polysaccharide chain, preferably constituted by chains of glucosidic units,

MC represents methylcarboxylic groups,

- B represents N-benzylmethylene carboxamide groups

- Su represents sulphate groups (sulphates of free hydroxyl functions carried by glucosidic units),

- a, b and c represent the degree of substitution (ds) respectively of groups MC, B and Su with

(i) a restriction greater than 0;

ii) b is such that

- b is greater than or equal to 0,3 and c is between 0,1 and 0,5,

- where b is strictly less than 0,3 and c corresponds to the following equation (1)

c> 8.5 b2-5.41 b + 0.86 (1) These dextran derivatives of formula (I) as well as their preparation process are described more generally in WO 99/29734. These dextran derivatives of formula (I) are commonly called DMCBSu and are considered to be copolymers consisting of R-OH and R-OX5 X subunits and may be a methylcarboxylic (MC), benzylamide (B) or sulfate (Su) group. Thus a dextratomethylcarboxylic (DMC) with a degree of substitution (ds) of 0.6 methylcarboxylic groups contains 0.6 substituted group (R-MC) and 2.4 hydroxyl groups (R-OH) per glucosidic unit.

In one embodiment, D has a molar mass comprised between 1,000 and 2,000,000 Da, and in one embodiment, less than 70,000 Da.

In one embodiment, dextran derivatives are selected from the compounds of formula (I) wherein b is greater than or equal to 0.35.

In this case, and according to the embodiment, the dextrate derivatives are selected from the compounds of formula (I) wherein a is from 0.5 to 0.8 and c is from 0.1 to 0.5.

In one embodiment, polysaccharides are selected from the group consisting of hyaluronans, alginates and oschitosans.

These different polysaccharides can be represented as follows:

<formula> formula see original document page 15 </formula> <formula> formula see original document page 16 </formula>

Hyaluronan

<formula> formula see original document page 16 </formula>

R = H, Dextran

R = CH 2 COOH or Η, Carboxymethyl Dextran

<formula> formula see original document page 16 </formula>

Chitosan

<formula> formula see original document page 16 </formula>

Alginate

The polysaccharide may have an average polymerization degree of between 10000.

In one embodiment, it has an average polymerization degree of between 10 and 5000.

In another embodiment, it has an average polymerization degree m of between 10 and 500 µm.

In one embodiment, the invention also relates to an amphiphilic polymer-PDGF complex characterized in that the hydrophobic group Hy is selected from the group consisting of fatty acids, fatty alcohols, fatty amines, benzyl amines, cholesterol derivatives and phenols.

In one embodiment the cholesterol derivative is colonic acid.

In another embodiment, phenol is alpha tocopherol.

In one embodiment, the amphiphilic polymer-PDGF complex according to the invention is reversible.

The polymers used are synthesized according to techniques well known in the art or purchased from suppliers such as Sigma-Aldrich, NOF corp. or CarboMer Inc.

PDGFs are selected from human recombinant PDGFs obtained according to techniques well known in the art or purchased from suppliers such as Gentaur (USA) or ResearchDiagnostic Inc. (USA).

Verification of both chemical and physical stabilization may be carried out notably by performing the following tests:

• a verification test of the amphiphilic polymer-PDGF complex according to the invention performed by gel electrophoresis (GelMobility Shift Assay)

• an enzyme degradation assay of an amphiphilic polymer-PDGF complex according to the invention performed by contacting a protease

A physical stabilization test of an amphiphilic polymer-PDGF complex according to the invention at a physiological pH performed by DSDS-P AGE.

The invention also relates to a polymer-amphiphilic PDGF complex characterized in that it meets the chemical and physical stabilization verification tests, namely a Gel Mobility Shift Assay verification test, the enzyme degradation velocity delay test. contact with a protease and the physiological pH stabilization test performed by SDS-Page.

Gel MobilityShift Assay's complex verification test based on the displacement of ions under the effect of an electric field. The anionic complexes migrate to the anode and the complexoscationic to the cathode. Following migration, proteins are transferred by capillary transfer over PVDF membrane and revealed by a protein-specific antibody recognized by a second peroxidase antibody. Protein alone does not migrate or migrate little, the complex protein of amphiphilic polymer migrates to the anode or cathode as a function of the overall charge of the complex.

The enzymatic degradation rate retardation test is based on verification of protein integrity after contacting the amphiphilic polymer-PDGF complex according to the invention with a protease. A protease solution (trypsin, trypsin ...) is added to the complex solution and kinetics are performed. The reaction is stopped by the addition of an enzyme-specific inhibitor (indole, benzamidine). Protein integrity is then analyzed by depolycrylamide gel electrophoresis (SDS-page).

The physical stabilization test of a PDGF is based on protein integrity check by comparing an amphiphilic polymer-PDGF complex solution according to the invention to a PDGF solution alone at pH 7.4 in terms of protein concentration in solution. These two solutions are placed on a shaking bank for 48 h at room temperature and then centrifuged. The concentration in PDGF in each solution is evaluated by SDS-Page.

The amphiphilic polymer-PDGF complex according to the invention is formed by placing in aqueous solution a PDGF and an amphiphilic polymer at a physiological pH without using an organic solvent capable of denaturing the protein. Formation of the amphiphilic polymer-PDGF complex is spontaneous and does not imply covalent bonding between the PDGF and the amphiphilic polymer. This association is made by weak bonds which are essentially hydrophobic interactions and ionic interactions.

The invention also relates to the process of preparing the amphiphilic polymer-PDGF complex according to the invention, characterized in that a PDGF and an amphiphilic polymer are contacted in physiological pH.

Other tests may be employed to verify the formation of the amphiphilic polymer-PDGF complex formation according to the invention.

• a maintenance test of the tertiary PDGF structure determined by circular dichroism,

A stability test of a PDGF in the amphiphilic polymer-PDGF complex according to the invention at physiological pH under stress. Stress may be a particular mode of agitation, the presence of salts, etc.

The invention also relates to an amphiphilic polymer-PDGF complex characterized in that the ratio PDGF / amphiphilic polymer is between 1/5 and 1 / 5,000.

In one embodiment, it is from 1/100 to 1/5000.

In another embodiment, it is comprised between 1/300 and 1/700.

The invention also relates to a characterized therapeutic composition wherein it comprises an amphiphilic polymer-PDGF complex according to the invention.

By therapeutic composition is meant a composition usable in human or veterinary medicine.

The pharmaceutical composition according to the invention is preferably a topically applied composition which may be in the form of a gel, cream, spray or paste, or adhesive dressing.

The nature of excipients which may be formulated as an amphiphilic polymer-PDGF complex according to the invention is selected according to their presentation according to the general knowledge of the pharmacist.

Thus, when the composition according to the invention is in the form of a gel, this is, for example, a gel made from polymers such as carboxymethyl celluloses (CMC), vinyl polymers, PEO_PPO-type polymers, polysaccharides, PEO, acrylamides, or acrylamide derivatives.

Other excipients may be used in this invention to adjust the formulation parameters as a pH adjusting buffer, such as isotonicity adjusting, preservatives such as methyl parahydroxybenzoate, propyl parahydroxy benzoate, m-cresol, or phenol or an antioxidant such as L-lysine hydrochloride.

According to the invention, the therapeutic composition is characterized in that it allows for administration of about 100 μg per ml PDGF.

The present invention also relates to the use of an amphiphilic polymer-PDGF complex according to the invention for the preparation of a healing therapeutic composition for topically treating ulcers.

It also relates to a method of therapeutic treatment for human or veterinary use characterized in that it consists in administering to the treatment site a therapeutic composition comprising the amphiphilic polymer-PDGF complex according to the invention.

EXAMPLESExample 1: PDGF-BB / DMCBSu Complex

Synthesis of Carbenzylamine-Modified Sulfated Carboxymethyl Dextran (DMCBSu)

The amphiphilic polymer is synthesized from a carboxymethylhextran having a degree of carboxymethyl substitution with acidic units of 1.0 and an average molar mass of 40,000 g / mol. Benzylamine is grafted onto the acids of this polymer according to a classical water copulation method in the presence of a water soluble carbodiimide. Substitution rate for benzylamine per saccharide unit is 0.4, determined by 1 H NMR. This polymer is then sulfated by an SO3 / pyridine complex. The degree of substitution in sulfate by units of acid is 0.3.

PDGF-BB / DMCBSu complex preparation

10 μl of a 0.1 mg / ml PDGF-BB solution is added to 90 μl of a 50 mg / ml DMCBSu solution. PDGF-BB and DMCBSu solutions are buffered to pH 7.4 and 300 mOsm. This solution is placed under gentle stirring for two hours at room temperature then stored at 4 ° C.

Verification of formation of a PDGF-BB / DMCBSu complex

10 μl of the above described PDGF-BB / DMCBSu complex solution is deposited on an agarose gel. The migration of the compounds is effected under the effect of an electric field (200 mA-4 hours). After amelioration, PDGF-BB is transferred onto a PVDF membrane overnight, then revealed by immunoblotting with goat anti-PDGF-BB antibody onto which HRP peroxidase-copulated decabra secondary antibodies will be fixed by a substrate ( 5-bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium).

The PDGF-BB / DMCBSu complex migrates towards the anode. Its negative charge is explained by a much richer composition in DMCBSu than in PDGF-BB. The control, consisting only of PDGF-BB, does not migrate.

Verification of the stability of the PDGF-complex solution

DMCBSu

10 μl of a 0.01 mg / ml PDGF-BB solution at pH 7.4 and 10 μl of the pH 7.4 PDGF-BB / DMCBSu complex solution described above are placed on a shaking bench for 48h at room temperature. After centrifugation, the concentration in PDGF-BB in each of the solutions is evaluated by SDS-Page. It appears that the concentration in PDGF-BB solution in the case of the PDGF-BB / DMCBSu complex did not change whereas that of the PDGF-BB solution alone decreased.

Verification of PDGF-BB trypsin protection in this PDGF-BB / DMCBSu complex

10 μl of the BMP-2 / DMCTrpOMe complex solution described above is poured into 90 μΐ of a 10 ng / ml trypsin solution at 37 ° C.

By a 10 μl sample withdrawal, the concentration inPDGF-BB is measured every 30 minutes by Elisa after stopping the enzymatic reaction by adding 10 μl of a 10 μg / ml indole solution.

This kinetics reveals that PDGF-BB alone is completely degraded by 1h30 whereas it is not in the PDGF-BB / DMCB Su complex.

Validation of the biological activity of PDGF-BB / DMCBSu complex

Human Dermal Fibroblast adult (HDFa) primary culture is performed at a temperature of 37 ° C in aMEM medium with 10% fetal calf serum (SVF) and 1% depenicillin-streptomycin in a moisture-saturated atmosphere. is enriched in CO2 (5%) The medium is renewed every 4 days A dilution of the cell suspension in the culture medium was then performed to seed culture plates at a density of 5,000 cells / wells for 96-well plates (Nunc company) .

For each cell batch, the stabilizing effect of PDGF-BB by the complex at different concentrations was verified by tritiated thymidine incorporation (5000 cells / wells at 100 μΐ). After 24 hours of deprivation, fibroblasts are stimulated by addition of PDGF-BB in different concentrations ranging from 0.1 to 100 ng / ml, in the presence or absence of amphiphilic polymer, at a concentration of 1 μ§ / ηι1. Tritiated datimidine incorporation is effected 18 hours after stimulation by PDGF-BB on or off the complex by adding a solution at 50 μΟί / ηύ, ie 0.5 μCi / wells, radioactivity is recovered in counting vials, the wells were rinsed with 100 μ 100 100 mM NaOH and the radioactivity is counted after the addition of 1 ml scintillation liquid (Zinsser Analytical) on an automatic calculator.

The results obtained are shown in the attached figure 1, in which the amount of tritiated thymidine incorporated by fibroblasts (in Dpm χ 103) is expressed as a function of PDGF-BB concentration in µg / ml.

The full stroke curve represents the results of the complex according to the invention at a concentration of 1 μg / ml in dextran derivative and the dotted curve curve results in the absence of dextran.

LED50 corresponds to PDGF-BB concentration to have 50% proliferation of human fibroblasts. The ratio of R is the ratio of ED50 calculated as follows:

R = ED50 (PDGF-BB) / ED50 (PDGF-BB + DMCBSu)

These results show that when PDGF-BB is used on its own, it is necessary to employ 6 μg / ml to obtain 50% proliferation, whereas when PDGF-BB is complexed by a derivex of formula (I), 2 μg / ml is sufficient. to reach 50% proliferation. Arelation R in this case is equal to 3.

Example 2: PDGF-BB / DMCTrpOMe Complex

Synthesis of tryptophan methyl ester carboxymethyl dextran (DMCTrpOMO)

The amphiphilic polymer is synthesized from carboxymethylhextran having a degree of carboxymethyl substitution with acidic units of 1.0 and an average molar mass of 40,000 g / mol. Tryptophan methylicode ester is grafted onto the acids of this polymer according to a classical water copulation method in the presence of water-soluble carbodiimide. The degree of substitution in tryptophan per saccharide unit is 0.3 determined by 1 H NMR.

PDGF-BB / DMCTrpOMe complex preparation

10 μl of a 0.1 mg / ml PDGF-BB solution is added to 90 μl of a 50 mg / ml DMCTrpOMe solution. PDGF-BB and DMCTrpOMe solutions are buffered at pH 7.4 and 300 mOsm. This solution is placed under gentle stirring two hours at room temperature then stored at 4 ° C.

Verification of formation of a PDGF-BB / DMCTrpOMe complex

10 μl of the above PDGF-BB / DMCTrpM complex solution described above is deposited on an agarose gel for an electrophoresis gel with an immunodevelopment. The migration of the compounds is effected under the effect of an electric field (200 mA-4 hours). After amelioration, PDGF-BB is transferred onto a PVDF membrane overnight, then revealed by immunoblotting with goat anti-PDGF-BB antibody onto which peroxidase-copulated decabra secondary antibodies will be attached (5). -bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium)

The PDGF-BB / DMCTrpOMe complex migrates toward the anode. Its negative charge is explained by a much richer composition in DMCTrpOMe than in PDGF-BB. I control, consisting only of PDGF-BB, does not migrate.

Stability check of PDGF-BB / DMCTrpOMe complex

10 μl of a 0.01 mg / ml PDGF-BB solution at pH 7.4 and 10 μl of the above described 7.4 PDGF-BB / DMCTrpOMe complex solution above are placed on a stirring bench for 48h at room temperature. The PDGF-BB concentration in each solution is evaluated by SDS-Page. Note that the concentration of PDGF-BB in solution for the PDGF-BB / DMCTrpOMe complex did not change while that of the PDGF-BB solution alone decreased.

Verification of PDGF-BB trypsin protection in this PDGF-BB / DMCTrpOMe complex

10 μl of the PDGF-BB / DMCTrp complex solution described above are incubated with 90μl of a 10 ng / mL Trypsin solution at 37 ° C. For a 10 μl withdrawal every 30 minutes, the structural integrity of PDGF-BB is assessed by depolycrylamide gel electrophoresis (SDS-Page) after stopping the enzymatic reaction by adding 10 μl of an indole solution at 10 μg / ml.

This kinetics reveals that PDGF-BB alone is completely degraded in 1h30 while it is not in the PDGF-B / DMCTrpOMe complex.

Example 3: PDGF-BB / CMCBSu Complex

Synthesis of Modified Sulfated Carboxymethyl Cellulose Pelabenzylamine (CMCBSu)

The amphiphilic polymer is synthesized from a carboxymethyl cellulose having a degree of carboxymethyl substitution with 1.2-carboxylic acid units and an average molar mass of 30000 g / mol. Benzylamine is grafted onto the acids of this polymer according to a classical water copulation method in the presence of a water soluble carbodiimide. Saccharide benzylamine substitution rate is 0.2 determined by 1 H NMR. Sulfation is performed in the presence of an S03-Pyridine complex, the degree of sulphate substitution is 0.30

PDGF-BB / CMCBSu complex preparation

10 μl of a 0.1 mg / ml PDGF-BB solution is added to 90 μl of a 50 mg / ml CMCB solution. PDGF-BB and deCMCBSu solutions are buffered at pH 7.4 and 300 mOsm. This solution is placed under gentle agitation two hours at room temperature then stored at 4 ° C.

Verification of formation of a PDGF-BB / CMCBSu complex

10 μl of the above described PDGF-BB / CMCBSu complex solution is deposited on an agarose gel for subtle electrophoresis with an immunodevelopment. The migration of the compounds is effected under the effect of an electric field (200 mA-4 hours). Following migration, PDGF-BB is transferred over a PVDF membrane overnight, then revealed by immunoblotting with goat anti-PDGF-BB antibody onto which goat anti-goat secondary antibodies to HRP peroxidase revealed by a substrate ( 5-bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium). The PDGF-BB / CMCBSu complex migrates towards the anode. Its negative charge is explained by a composition richer in CMCBSu than in PDGF-BB. The control, consisting only of PDGF-BB, does not migrate.

Stability check of PDGF-BB / CMCBSu complex

10 μl of a 0.01 mg / ml PDGF-BB solution at pH 7.4 and 10 μl of the 7.4 7.4 PDGF-BB / CMCBSu complex solution described above are placed on a stirring bench for 48h at room temperature. After centrifugation, the concentration in PDGF-BB in each of the solutions is evaluated by SDS-Page. It is noted that the concentration in PDGF-BB solution in case of the PDGF-BB / CMCBSu complex is not changed whereas that of PDGF-BB solution alone decreased.

Verification of PDGF-BB trypsin protection in this PDGF-BB / CMCBSu complex

10 μl of the above described PDGF-BB / CMCBSu complex solution are incubated with 90μΙ of a 10 ng / mL Trypsin solution at 370C. For a 10 μΐ withdrawal every 30 minutes, the structural integrity of the PDGF-BB is assessed by polyacrylamide gel electrophoresis (SDS-Page) after stopping the enzymatic reaction by adding 10 μΐ of an indole solution at 10 μg / ml. .

This kinetics reveals that PDGF-BB alone is completely degraded by 1h30 while it is not in the PDGF-BB / DMCBSu complex.

Counterexample 1: PDGF-BB / CMCB complex

Synthesis of benzylamine modified carboxymethyl cellulose (CMCB)

The amphiphilic polymer is synthesized from a carboxymethyl cellulose having a degree of carboxymethyl substitution saccharide perunit of 1.2 and an average molar mass of 30000 g / mol. Abenzylamine is grafted onto the acids of this polymer according to a classical water copulation method in the presence of a water-soluble carbodiimide. The degree of substitution in benzylamine by saccharide unit is 0.2 determined by1 H NMR.

PDGF-BB / CMCB complex preparation

10 μl of a 0.1 mg / ml PDGF-BB solution is added to 90 μl of a 50 mg / ml CMCB solution. PDGF-BB and deCMCB solutions are buffered at pH 7.4 and 300 mOsm. This solution is placed under gentle agitation two hours at room temperature then stored at 4 ° C.

Verification of PDGF-BB / CMCBμl complex non-formation of the above described PDGF-BB / CMCB complex solution is deposited on an agarose gel for a superlative electrophoresis with an immunological development. The migration of the compounds is effected under the effect of an electric field (200 mA-4 hours). After migration, oPDGF-BB is transferred over a PVDF membrane overnight, then revealed by immunoblotting with goat anti-PDGF-BB antibodies onto which goat anti-IgG secondary antibodies are attached to HRP peroxidase revealed by a substrate ( 5-bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium).

The disclosure does not show migration of PDGF-BB alone with amphiphilic opolymer. There was no complex formation between CMCB and oPDGF-BB.

Claims (26)

1. Water-soluble physically stable, amphiphilic polymer-PDGF complex, characterized in that the amphiphilic polymers consist of a hydrophilic skeletopolymer functionalized by hydrophobic substituents and hydrophilic groups according to the following general formula. <formula> formula see original document page • R, R 'being a bond or chain comprising from 1 to 18 carbons, optionally branched and / or unsaturated comprising one or more heteroatoms, such as O, N and / or S, ReR' being identical or different from each other • F, F1 being an ester, a thioester, an amide, a carbonate, a carbamate, an ether, a thioether, an amine, FeF 'being identical or different from each other • X being a hydrophilic grouping which may be: o a carboxylate a sulfate a sulfonate a phosphate a phosphonate being a hydrophilic group may be a sulfate a sulfonate a phosphate a phosphonate Nozzle may be a C8 to C30 straight or branched alkyl, optionally unsaturated and / or containing one or more heteroatoms such as O, N or S, o a C8 to C8 straight or branched alkylaryl or arylalkyl, optionally unsaturated and / or containing optionally a heteroatom, a possibly unsaturated C8 to C30 polycyclo, η and o are between 1 and 3, h represents the molar fraction of hydrophobic motif with respect to a monomer unit comprised between 0.01 and 0.5, χ represents the molar fraction of hydrophilic groups with respect to a monomeric unit comprised between 0 and 2.0, y represents the molar fraction of hydrophilic groups with respect to a monomeric unit comprised between 0 and 0.5, - PDGF is selected from the group consisting of PDGFs (Platelet -derivated growth factors). Complex according to claim 1, characterized in that PDGF is selected from the group consisting of human recombinant PDGFs having two B-chains (rhPDGF-BB). Complex according to any one of the preceding claims, characterized in that the polymer is selected from polymers whose substituents are statistically divided. Complex according to any one of the preceding claims, characterized in that the amphiphilic polymer is selected from polyamino acids. Complex according to claim 4, characterized in that the polyamino acids are selected from the group consisting of polyglutamates or polyaspartates.-1 and 2, characterized in that, to generate the order of rotation induced by the exit consignment and to perform the submission, the means that are formed from a theory called modal command are used. Complex according to claim 5, characterized in that the polyamino acids are homopolyglutamates. Complex according to claim 6, characterized in that the polyamino acids are homopolypartates. Complex according to claim 7, characterized in that the polyamino acids are aspartate eglutamate copolymers. Complex according to one of Claims 1 to 5, characterized in that the polymer is selected from polysaccharides. Complex according to claim 9, characterized in that the polysaccharides are selected from the group consisting of hyaluronans, alginates, chitosones, galacturonans, achondroitin sulfate, dextrates, celluloses. Complex according to Claim 10, characterized in that the group of celluloses is made up of acid-functionalized celluloses such as carboxymethyl cellulose. The complex of claim 10, characterized in that the group of dextrans consists of dextrans functionalized by the acids as carboxymethyl dextran. Complex according to claim 9, characterized in that the polysaccharides are selected from the group consisting of hyaluronans, alginates, chitosans. Complex according to claim 9, characterized in that the polysaccharides are selected from the group consisting of soluble dextran derivatives by answering the following formula (I): whereinD represents a polysaccharide chain, preferably constituted by chains of glucosidic units, - MC represents methylcarboxylic groups, - B represents N-benzylmethylene carboxamide groups - Su represents sulfate groups (sulfation of free hydroxyl functions carried by the glucosidic units), - a, b and c represent the degree of substitution (ds) of clusters MC, B and Su respectively i) a restriction greater than 0, ii) b is such that b is greater than or equal to 0.3 and c is between 0.1 and 0.5, - where b is strictly less than 0.3 and c corresponds to equation (1) following> 8.5 b2-5.41 b + 0.86 (1). Complex according to any one of the preceding claims, characterized in that the hydrophobic group Hy is selected from the group consisting of fatty acids, fatty alcohols, fatty amines, benzyl amines, cholesterol derivatives and phenols. Complex according to any one of the preceding claims, characterized in that the formation of the amphiphilic polymer-PDGF complex is reversible. Complex according to any one of the preceding claims, characterized in that it meets the tests for chemical and physical stabilization. A complex according to claim 17, characterized in that chemical and physical stabilization verification tests are selected from the group consisting of a Gel Mobility Shift Assay verification test, a decrease in contact enzymatic degradation test. with a protease and a physiological apH physical stabilization test performed by SDS-PAGE. Complex according to any one of the preceding claims, characterized in that the ratio PDGF / amphiphilic polymer is between 1/5 and 1 / 5,000. Complex according to any one of the preceding claims, characterized in that the ratio PDGF / amphiphilic polymer is between 1/100 and 1 / 5,000. Complex according to any one of the preceding claims, characterized in that the ratio PDGF / amphiphilic polymer is between 1/300 and 1/700. Process for preparing the amphiphilic polymer-PDGF complex as defined in any one of the preceding claims, characterized in that this amphiphilic polymer-PDGF complex is prepared in aqueous medium and in the absence of the organic solvent capable of denaturing the protein. Therapeutic composition characterized in that it comprises an amphiphilic polymer-PDGF complex as defined in any one of claims 1 to 21. Therapeutic composition according to claim 20, characterized in that it allows 100 µg per ml of PDGF to be administered. Use of an amphiphilic polymer-PDGF complex as defined in any one of claims 1 to 21, characterized in that it is for the preparation of a scarring therapeutic composition for topically treating ulcers. Use of a therapeutic composition comprising an amphiphilic polymer-PDGF complex as defined in any one of claims 1 to 21, characterized in that it is for the manufacture of a medicament for human or veterinary therapeutic treatment.